Molecular hydrogen (H2) is typically produced by steam reforming of methane, and platinum is the most commonly used catalyst for hydrogen production. Due to utilization of fossil fuels as a source of methane, as well as the expense, limited availability, sensitivity to poisoning, and bioincompatibility of the catalyst, it is not likely to be utilized in economical energy conversion systems (Bharadwaj and Schmidt. 1995. Fuel Processing Technology 42:109-127, Ghenciu. 2002. Current Opinion in Solid State & Materials Science 6:389-399). However, in 2003 President Bush in the State of the Union Address proposed the Hydrogen Fuel Initiative, the goal of which was to develop new technologies for production and utilization of H2 as a potential source of energy to replace fossil fuels. In microorganisms, the molecular machine responsible for the biological uptake and evolution of hydrogen is an enzyme known as hydrogenase. Hydrogenase catalyzes the simplest of chemical reactions, the interconversion of the neutral molecule H2 and its elementary constituents, two protons and two electrons (Eqn. 1).2H++2e−←→H2  (1)Ironically, however, while the reaction that they catalyze is simple, hydrogenase enzymes are multimeric proteins and typically are sensitive to air (oxygen). This has to-date precluded the facile production of a recombinant form of the major class of hydrogenase, the so-called ‘nickel-iron’ (NiFe) type.
Hydrogenases are found in representatives of most microbial genera, as well as some unicellular eukaryotes (Adams et al. 1980. Biochim Biophys Acta 594:105-76; Cammack et al. 2001. Hydrogen as a fuel: learning from nature. Taylor & Francis, London, New York; Friedrich and Schwartz. 1993. Annual Review of Microbiology 47:351-383; Przybyla et al. 1992. FEMS Microbiology Reviews 88:109-135, Vignais et al. 2001. FEMS Microbiology Reviews 25:455-501). The enzyme allows many microorganisms to use H2 gas as a source of low potential reductant (H2/H+, Eo′=−420 mV), either for carbon fixation or as a source of energy. In aerobic environments, H2 oxidation can be coupled via membrane electron transport to the reduction of oxygen (O2/H2O, Eo′=+820 my). There are a variety of electron acceptors that can be coupled to anaerobic H2 oxidation, including carbon dioxide, which can be reduced to either methane (by methanogens) or acetate (by acetogens), and sulfate and ferric-iron, which are reduced to sulfide and ferrous iron, respectively. On the other hand, microorganisms that produce H2 during growth are widespread in anaerobic environments. The production of H2 is used as a mechanism to dispose of the excess reductant that is generated during the oxidation of organic material. These fermentative organisms conserve energy by chemical synthesis (substrate level phosphorylation) independent of the means by which they dispose of reductant (be it as H2 or as a reduced organic compound such as ethanol). However, it was recently discovered that some organisms are able to conserve energy directly from the production of H2 by a novel respiratory mechanism (Sapra et al. 2003. Proc Natl Acad Sci USA 100:7545-50).
Two major types of hydrogenase are known: the nickel-iron (NiFe) and the iron-only (Fe) enzymes (Adams. 1990. Biochimica Et Biophysica Acta 1020:115-145; Albracht. 1994. Biochimica Et Biophysica Acta-Bioenergetics 1188:167-204), which are unrelated phylogenetically (Meyer, J. 2007. Cellular and Molecular Life Sciences 64:1063-1084; Vignais et al. 2001. FEMS Microbiology Reviews 25:455-501). The iron-only type is found in only a few types of anaerobic bacteria and some photosynthetic algae, but they have been extensively studied. This includes structural characterization (Chen et al. 2002. Biochemistry 41:2036-2043; Nicolet et al. 2001. Journal of the American Chemical Society 123:1596-1601; Nicolet et al. 2000. Trends in Biochemical Sciences 25:138-143; Nicolet et al. 1999. Structure with Folding & Design 7:13-23; Peters et al. 1998. Science 282:1853-1858) including potential active site models (Boyke et al. 2004. Journal of the American Chemical Society 126:15151-15160; Tye et al. 2006. Inorg Chem 45:1552-9; Zilberman et al. 2007. Inorg Chem 46:1153-61), and recently insights have been provided into their biosynthesis (Mishra et al. 2004. Biochemical and Biophysical Research Communications 324:679-685; Posewitz et al. 2004. Journal of Biological Chemistry 279:25711-25720), as well there are some recent successful attempts to make recombinant forms of these enzymes (King et al. 2006. J Bacteriol 188:2163-72).
The majority of microorganisms that metabolize H2, however, contain NiFe-hydrogenases, an example of which is the cytoplasmic NiFe hydrogenase I of the hyperthermophilic archaeon, Pyrococcus furiosus, which grows optimally at 100° C. (Fiala and Stetter. 1986. Archives of Microbiology 145:56-61, Verhagen et al. 2001. Hyperthermophilic Enzymes, Pt A 330:25-30). The NiFe-hydrogenases have also been extensively characterized over the last 40 years, and several crystal structures are available (Garcin et al. 1998. Biochemical Society Transactions 26:396-401, Higuchi. 1999. Structure 7:549-56, Volbeda and Fontecilla-Camps. 2003. Dalton Transactions: 4030-4038, Volbeda et al. 1996. Journal of the American Chemical Society 118:12989-12996). They all are made up of at least two subunits, one of which contains the NiFe-catalytic site, while the other contains three iron-sulfur (FeS) clusters. These clusters serve to shuttle electrons from the electron donor to the enzyme to and from the NiFe site in the catalytic subunit. The Ni atom is bound to four cysteinyl residues of this subunit, two of which are near the N-terminus and two near the C-terminus. Two of the four Cys bind a single Fe atom, which is also coordinated, remarkably, by one carbon monoxide (CO) and two cyanide (CN) ligands (Bagley et al. 1995. Biochemistry 34:5527-5535, Happe et al. 1997. Nature 385:126-126, Pierik et al. 1999. Journal of Biological Chemistry 274:3331-3337). These diatomic ligands serve to activate the iron atom (maintaining it in the low spin state) thereby facilitating catalysis. Interestingly, such ligands are also found at the active site of the iron-only hydrogenases (Nicolet et al. 2002. J Inorg Biochem 91:1-8), as well as the mononuclear iron site of a third type of hydrogenase found in a very limited number of archaea (Lyon et al. 2004. Journal of the American Chemical Society 126:14239-14248), an example of convergent evolution toward a similar function.
The hydrogenase of P. furiosus is of particular interest for additional reasons. First, it is obtained from an organism that grows optimally at 100° C. and has been shown to be an exceedingly robust and thermostable enzyme (Bryant and Adams. 1989. J Biol Chem 264:5070-9; Ma and Adams. 2001. Methods Enzymol 331:208-16). Second, in in vitro assays, the enzyme has been shown to be able to generate hydrogen gas by oxidizing NADPH in a reversible reaction (Ma and Adams. 2001. Methods Enzymol 331:208-16; Ma et al. 2000. J Bacteriol 182:1864-71; Ma et al. 1994. FEMS Microbiology Letters 122:245-250), which is a very rare property among the hydrogenases that have been characterized to date. Consequently, the reversible P. furiosus enzyme has utility in generating reductants such as NADPH. Likewise, the P. furiosus enzyme has utility in hydrogen production systems in which carbohydrates are oxidized to generate NADPH, which in turn can be converted to hydrogen gas by the hydrogenase. The production of hydrogen from glucose in an in vitro cell-free system using purified enzymes was first demonstrated over a decade ago (Woodward et al. 1996. Nat Biotechnol 14:872-4). This work was very recently extended in which the conversion of starch to hydrogen was described using an in vitro cell-free system made up of thirteen different enzymes (Zhang et al. 2007. PLoS ONE 2:e456). Twelve of the enzymes are used to oxidize starch and generate carbon dioxide and NADPH, and the thirteenth, P. furiosus hydrogenase, oxidizes NADPH and produces hydrogen gas. In this system, the hydrogenase was purified from P. furiosus biomass (Ma and Adams. 2001. Methods Enzymol 331:208-16) since a recombinant form of this enzyme was not available.